Epigenetic processes interpret the genomic sequences in a cell-type and extracellular environment-dictated mode. Epigenetic information is encoded through covalent modifications of histones and DNA, nucleosome position and substitution by histone variants. Epigenetics is one of the most intensively studied fields of biology today and represents a new paradigm for the pathophysiology, diagnosis and treatment of diseases. To advance epigenetic studies, we have introduced a novel chromatin immunoprecipitation platform, Matrix ChIP, that utilizes surface-immobilized antibodies in a 96-well plate, where the entire procedure from chromatin precipitation to PCR-ready DNA purification is done on the same plate without sample transfers. This high- throughput method allows for parallel profiling of an order of magnitude greater number of chromatin and transcription events than was previously possible. Matrix ChIP is more sensitive than traditional approach to detect DNA-protein interactions of less abundant proteins such as chromatin modifiers. ChIP assay consists of three separate steps: (1) sample harvesting/chromatin fragmentation, (2) immunoprecipitation and (3) DNA purification and analysis (qPCR/sequencing). Although in the Matrix ChIP, steps (2) and (3) are greatly simplified allowing one to run hundreds of assays at a time, sample harvesting/chromatin fragmentation is done in test tubes with low efficiency and high labor intensity. This project proposes an innovative multidisciplinary approach (Dr. Matula, ultrasound/engineering, Dr. Denisenko, molecular biology, and Dr. Bomsztyk, chromatin biology/biotechnology/instrumentation) to develop a platform, PIXUL-ChIP, that will integrate highly efficient sample harvesting and chromatin shearing with immunoprecipitation in microplates for high-throughput epigenetic analysis. The following aims are proposed. Aim#1. To build a pixelated ultrasound (PIXUL) processor for high-throughput fast chromatin shearing. Aim#2. To combine sample harvesting and chromatin shearing with immunoprecipitation into an integrated microplate platform for epigenetic analysis, PIXUL-ChIP. Aim#3. To test PIXUL-ChIP as a platform for studies of epigenetic changes at selected gene loci associated with drug-mediated human embryonic stem cell (hESC) renewal and differentiation. Epigenetics is a fast growing field, but nonetheless, there are significant unmet needs that require further technological advances to facilitate exploration of epigenetic processes and exploit this knowledge in translational research and clinical applications. A highly efficient technology, PIXUL-ChIP will provide powerful means to simultaneously study multiple chromatin modifications and modifiers at gene loci in a wide range of systems, and as such will be a valuable platform for studying epigenetic processes, discovering epigenetic biomarkers, and testing novel drugs and their combinations.